Thrombin is a naturally occurring protein which has several bioregulatory roles [J. W. Fenton, II, "Thrombin Bioregulatory Functions", Adv. Clin. Enzymol., 6, pp. 186-93 (1988)]. In addition to being intimately involved in blood coagulation, thrombin is also known to play a role in platelet and endothelial cell activation, smooth muscle cell and neuroblast proliferation and bone resorption. Thrombin exerts its various biological effects through one of two mechanisms. The first is purely enzymatic, involving only the catalytic activity of thrombin. This is the mechanism by which thrombin converts fibrinogen to fibrin, the final step in the coagulation cascade.
The second mechanism of thrombin action is mediated through a cell surface receptor. This receptor has recently been cloned [T.-K. H. Vu et al., "Molecular Cloning of a Functional Thrombin Receptor Reveals a Novel Proteolytic Mechanism of Receptor Activation", Cell, 64 pp 1057-68 (1991)]. It is believed that thrombin activates cells via its receptor by first associating with the hirudin-like extracellular domain of the receptor (receptor amino acids 52-69) [L.-W. Liu et al., "The Region of the Thrombin Receptor Resembling Hirudin Binds to Thrombin and Alters Enzyme Specificity", J Biol Chem., 266, pp. 16977-80 (1991)]. Thrombin then cleaves off a 41 amino acid peptide from its receptor's extracellularly located N-terminus, exposing a new N-terminus on the receptor. The newly exposed N-terminus then interacts with another, distant part of the receptor, resulting in activation. It is believed that activation may ultimately involve a secondary messenger, such as a kinase or cAMP, which is triggered by the cleaved receptor.
It has previously been shown that a peptide corresponding to the first 14 amino acids of the newly exposed N-terminus of the thrombin receptor (referred to as "the tethered ligand peptide") can activate the thrombin receptor in the absence of thrombin. This activation does not require the presence of an extracellular anionic domain in the receptor [T.-K. H. Vu et al., Supra].
The receptor-driven mechanism is responsible for thrombin-induced platelet aggregation and release reactions. Such reactions initiate arterial clot formation. In addition, the receptor mechanism is thought to be responsible for thrombin-induced endothelial cell activation. This activation stimulates the synthesis of platelet activating factor (PAF) in these cells [S. Prescott et al., "Human Endothelial Cells in Culture Produce Platelet-Activating Factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) When Stimulated With Thrombin", Proc Natl. Acad. Sci. USA, 81, pp. 3534-38 (1984)]. PAF is exposed on the surface of endothelial cells and serves as a ligand for neutrophil adhesion and subsequent degranulation, which results in inflammation [G. M. Vercolletti et al., "Platelet-Activating Factor Primes Neutrophil Responses to Agonists: Role in Promoting Neutrophil-Mediated Endothelial Damage", Blood, 71 pp. 1100-07 (1988)]. Alternatively, thrombin may promote inflammation by altering endothelial cells to produce increased vascular permeability which can lead to edema [P J. Del Vecchio et al., "Endothelial Monolayer Permeability To Macromolecules", Fed. Proc., 46, pp. 2511-15 (1987)], a process that also may involve the thrombin receptor.
Smooth muscle cells are also known to express thrombin receptors on their surface [D. T. Hung et al., "Thrombin-induced Events in Non-Platelet Cells Are Mediated by the Unique Proteolytic Mechanism Established for the Cloned Platelet Thrombin Receptor, J. Cell Biol., 116, pp. 827-32 (1992)]. When thrombin interacts with these cells, a proliferative response is generated. This proliferation of smooth muscle cells may contribute to restenosis following balloon angioplasty and subsequent coronary failure [A. J. B. Brady et al., "Angioplasty and Restenosis", Brit. Med. J., 303, pp. 729-30 (1991)]. Other cells which have thrombin receptors are fibroblasts, neuroblasts and osteoclasts. For each of these cells, thrombin has been shown to be responsible for some bioregulatory function.
For example, thrombin has been implicated in neurodegenerative diseases because of its ability to cause neurite retraction [D. Gurwitz et al., "Thrombin Modulates and Reverses Neuroblastoma Neurite Outgrowth", Proc Natl Acad Sci USA, 85, pp 3440-44 (1988)]. And thrombin has been hypothesized to play a role in osteoporosis due to its ability to stimulate bone resorption by osteoclasts [D. N. Tatakis et al., "Thrombin Effects On Osteoblastic Cells -II. Structure-Function Relationships", Biochem. Biophys. Res. Comm., 174, pp. 181-88 (1991)].
Therefore, the ability to regulate the in vivo activity of thrombin has many significant clinical implications. More importantly, the ability to selectively inhibit thrombin's receptor-driven functions without affecting fibrin formation is highly desirable so that bleeding will be reduced or eliminated as a potential side effect of treatment.
Compounds which inhibit thrombin directly may inhibit some or all of thrombin's functions to one degree or another. All active site inhibitors of thrombin inhibit both enzymatic and receptor-mediated functions of the molecule. Surprisingly, other thrombin inhibitors which do not bind to the active site can also inhibit both mechanisms. For example, peptides modelled on the C-terminal amino acid sequence of hirudin, a naturally occurring anticoagulant isolated from leeches, inhibit both fibrinogen cleavage, as well as thrombin-induced platelet and endothelial cell activation [U.S. patent application Ser. No. 677,609 now U.S. Pat. No. 5,256,559].
Other compounds are capable of selectively inhibiting thrombin's activities, but these compounds do not inhibit thrombin or its receptor directly and therefore are less specific. For example, heparin, which complexes with and activates antithrombin III, affects fibrin clot formation without affecting platelet-dependent thrombosis. But certain patients cannot be treated with heparin because of circulating antibodies against this compound. Also, heparin is known to cause bleeding complications. Arg-Gly-Asp-containing peptides inhibit platelet activation, without inhibiting fibrin formation. However, these peptides are general antiplatelet agents and are not specific for thrombin-induced platelet activation. These peptides work by competitively binding to the platelet surface protein, glycoprotein IIb/IIIa.
Thus, there is a great need for a direct and selective antagonist of thrombin's functions. Specifically, a compound which can inhibit thrombin's receptor-mediated functions without affecting fibrin-mediated clotting would have great utility. Such a compound would be particularly useful in preventing restenosis following angioplasty--a phenomenon linked to thrombin-induced smooth muscle cell proliferation [J. N. Wilcox, "Thrombin and Other Potential Mechanisms Underlying Restenosis", Circulation, 84, pp 432-35 (1991)]. Thrombin receptor antagonists would also be useful in treating or preventing arterial thrombosis without blocking fibrin formation.